We have recently identified a novel protein secretion pathway (proposed to be a Type IX Secretion System;T9SS) employed by periodontopathogens, including Porphyromonas gingivalis, Prevotella intermedia, and Tannerella forsythia to secrete a number of virulence factors. Progress on our current parent R01 grant has revealed the presence of 3 to 5 new essential components participating in the gingipains secretion pathway utilized by P. gingivalis. Interestingly, a large number of the newly identified genes along with those already reported apparently encode for putative integral outer membrane (OM) beta-barrel proteins. The requirement of several beta-barrel OM proteins for secretion is unique for T9SS and sets this OM translocation apparatus apart from eight other known secretion system in Gram-negative bacteria. In this application, responding to NOT-OD-09-058 (Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications) we will: (i) verify transmembrane topology of integral OM beta-barrel proteins;(ii) investigate the subcellular location of other proteins essential for gingipains secretion;(iii) determine the secretome of P. gingivalis, P. intermedia and T. forsythia which is dependent on T9SS, (iv) perform an initial characterization on proteases of T. forsythia bearing unique domain structure, which are most likely secreted via T9SS. PUBLIC HEALTH RELEVANCE: The results of these proposed studies should yield important information on unique proteases of T. forsythia which may function as virulence factors and shade light on a novel pathway for the protein secretion by P. gingivalis, T. forsythia and P. intermedia. This insight may enable the design of antibodies or chemical compounds to block the export of multiple virulence factors from major periodontopathogens, which in the long term, may revolutionize treatment and prophylaxis of periodontal disease.